Inflammation is a local response to tissue damage or foreign material which is designed to isolate and/or destroy injured tissues and foreign substances. Uncontrolled inflammatory responses may irreparably damage host tissue, as in the chronic inflammation of rheumatoid arthritis or lead to multiple organ failure and death as in the systemic inflammation of septic shock.
Eicosanoids are metabolic products of the essential fatty acid, arachadonic acid and are known to play a central role in the inflammatory response. Arachadonic acid is an integral component of cell membranes where it is commonly found as an sn-2 acyl or akyl ester of 3-sn-phosphoglycerides. Phospholipase A2 (PLA.sub.2) is a class of enzymes that specifically catalyze the hydrolysis of the sn-2 acyl or alkyl ester of phosphoglycerides, producing equimolar quantities of lysophospholipids and free fatty acids. The PLA.sub.2 catalyzed hydrolysis of one such membrane phospholipid, alkyl-arachidonyl-glycerophosphatidylcholine, yields free arachidonic acid and lyso-platelet activating factor (lyso-PAF), the precursor of PAF, in equimolar amounts. Since the availability of free arachidonic acid is rate limiting for eicosanoid synthesis, the pro-inflammatory role of PLA.sub.2 is thought to be the consequence of its role in arachadonic acid metabolism.
Three mammalian PLA.sub.2 enzymes are known. Pancreatic PLA.sub.2 is related to the type I PLA.sub.2 from the venom of Elapidae and Hydrophide both in primary and tertiary structure. A digestive enzyme, synthesized as a proenzyme in the pancreas, it is unlikely to play a central role in inflammatory conditions.
Two non-pancreatic mammalian enzymes have been described. One is a high molecular weight intracellular enzyme. The other is a soluble enzyme, sPLA.sub.2, and is of particular interest because it has been isolated from inflammatory exudates, such as synovial fluid (Seilhamer et al. (1989) J. Biol. Chem. 264: 5335-5338) and from platelets (Kramer et al. (1989) J. Bio. Chem. 264: 5768-5775). sPLA.sub.2 is known by several equivalent names, including secretory phospholipase A.sub.2, soluble phospholipase A.sub.2 and synovial phospholipase A2, all of which can be used interchangeably. This enzyme, which has been sequenced, has a molecular weight of 14 kD, a pI&gt;10 and is homologous to type II PLA.sub.2 from the venoms of Crotalidae and Viperidac (Kramer et al. (1989) supra; Scott et al. (1991) Science 254: 1007; Wery et al. (1991) Nature 352: 79-82).
The involvement of sPLA.sub.2 in the inflammatory response is supported by two types of data. First, elevated levels of serum PLA.sub.2 activity have been observed in diseases such as endotoxemia (Vadas and Hay (1983) Can. J. Physiol. Pharmacol. 61: 561-566), sepsis (Vadas (1984) J. Lab. Clin. Med. 104: 873-881; Nevalainen et al. (1992) Clin. Chem. 38: 1824-1829), rheumatoid arthritis (Pruzanski et al. (1988) J. Rheumatol. 15: 1351-1355), pancreatitis (Nevalainen et al. (1993) Gut 34: 1133-1136) and uremia (Costello et al. (1990) Clin. Chem. 36: 198-200) and in some studies correlate with severity and outcome. One of the best documented cases is acute pancreatitis in which sPLA.sub.2 immuno-reactive activity, but not pancreatic PLA.sub.2 immuno-reactive activity, is correlated with serum PLA.sub.2 enzymatic activity. The duration of the elevated levels is longer for the more severe necrotizing pancreatitis than the less severe oedmatous form (Nevalainen et al. (1993) supra).
Second, in both animal and tissue models (Snyder et al. (1993) J. Pharmacol. and Therapeutics 266: 1147-1155), the introduction of sPLA.sub.2 results in an inflammatory like response.
The isolation of specific antagonists to sPLA.sub.2 would have multiple uses. First, sPLA.sub.2 ligands would provide a useful tool for defining the enzyme's role in inflammatory responses and in diagnosing various inflammatory conditions. Second, sPLA.sub.2 antagonists would be useful as an anti-inflammatory therapeutic. Given the cationic nature of sPLA.sub.2 and the specificity of high affinity oligonucleotide ligands, SELEX technology is well suited for the isolation of sPLA.sub.2 antagonists which would not cross react with pancreatic or high molecular weight PLA2s. The present invention demonstrates the successful isolation of high-affinity oligonucleotide antagonists to sPLA.sub.2.